The Preparation Of Rat Cirrhosis Models Accompanied With Ascites And Their Use In Pharmacodynamics Evaluation Test For RHSA And Pd-CSA

Liver fibrogenesis is a dynamic process involving complex cellular andmolecular mechanisms, resulting in the chronic activation of the tissue repairmechanisms that follow repeated hepatic injury. One of the first steps in tissue repairis the recruitment of inflammatory cells in order to neutralize possible infectiousagents and to remove necrotic tissue. The onset of ascites in liver cirrhosis, a sign ofentry into the noncompensatory stage, is critical to the prognosis of the disease. Themechanism of its onset is quite complex and includes plasma colloid osmotic pressurereduction due to hypoalbuminemia.Both the research of liver cirrhosis pathomechanism and curative medicinesstudy greatly depend on the preparation of animal liver damage models. It is welldocumented that cirrhosis of the liver can be produced in rats by dosing them twiceweekly with carbon tetrachloride (CCl₄). However this method has serious drawbacks.It requires a long time, many animals die intercurrently, and a proportion and of therats that survive do not develop cirrhosis even after prolonged dosage, so the problemof producing a stable, homogeneous and consistent yield of sever cirrhosis withascites remains.The acute toxicity of CCl₄is dependent upon its metabolism by microsomalhydroxylating enzyme systems of the liver, and toxicity is therefore enhanced byphenobarbitone and other inducers of these systems, such as alcohol. The followingexperiments are set up to see if simultaneous treatment with CCl₄, Phenobarbitoneand alcohol would give a more rapid and reliable yield of cirrhosis of the liver. On thebasis of the improvement research we use the stable liver cirrhosis rat models to carryout the pharmacodynamics evaluation test of serum albumin.(1) The research and preparation on an improvement and optimization methodof CCl₄induced liver cirrhosis rats model accompanied with ascitesTo set a stable, homogeneous, well-tolerated, efficient protocol to induce ratcirrhosis models accompanied with ascites, we used an improved CCl₄combined withPhenobarbital sodium and alcohol method, and established a basis for our next step work of serum albumin effectiveness evaluation test.Ninety male Wistar rats were divided into five groups randomly: blank controlgroup contained10rats, and CCl₄group, CCl₄+phenobarbital sodium group,CCl₄+alcohol group and CCl₄+phenobarbital sodium+alcohol group contained20ratsrespectively as induce model groups. Control group was given normal drinking waterand injected with olive oil intraperitoneally; Rats in model groups were givendifferent drinking water and an intraperitoneal injection of CCl₄in a2:3mixture witholive oil. The drugs given intraperitoneally were all at a dose of2ml/kg body weight,twice weekly for12weeks. We further measured the body weight and abdominalcircumference, and tested the biochemical liver function index as well as the colloidosmotic pressure (COP) to evaluate the difference process in these groups. At the endof the induce operation the randomly selected rats in each group were executed, and asection of right liver lobe was obtained to examined by histopathological method. Thedata statistical analysis was performed.Cirrhosis accompanied with ascites developed in all model groups between8and12weeks. However, at the end of the eighth week, rats in CCl₄+phenobarbitalsodium+alcohol group were firstly detected to correspond with the model standards;CCl₄+phenobarbital sodium group and CCl₄+alcohol group were detected to havepositive model signs at the ninth week; only at the tenth week a few rats appeared tohave ascites in CCl₄group. All the induce model groups had significantly differencesabout the biochemical liver function index, COP and body weight than those rats incontrol group, however the most significant differences were observed inCCl₄+phenobarbital sodium+alcohol group. The model rate in model groups was65%,75%,75%and80%respectively. Histological examination showed that all the modelgroups developed liver cirrhosis with typical pseudolobule formation. The use ofcarbon tetrachloride associated with Phenobarbital sodium and alcohol presented astable, homogeneous, well-tolerated, effective method to induce cirrhosis rat modelswith ascites; the model rate had a good advantage than other transitional methods.We utilized the improved method to prepare90CCl₄induced rat liver cirrhosismodels and use them in the pharmacodynamics evaluation test of serum albumin(rHSA and pd-CSA).(2) Effectiveness of rHSA in the treatment of ascites in rat liver cirrhosis modelsAlbumin is the most abundant protein in plasma and is principally known for itsrole in the regulation of vascular oncotic pressure. Therapeutic human serum albumin (HSA) is an important plasma-derived product used for treatment of hypoalbuminenia.Today, considerations of ethics, safety and stability of supply indicate a need for adomestic supply of plasma fraction fraction preparations through blood donation.However albumin is used in large quantities, so supply of the raw material blood fromdomestic sources only would be difficult. Against this background, using recombinantDNA technology to develop human HSA is a good program to fill the albuminshortfall.In the second part of this study, to determine the effectiveness of rHSA, whichwas manufactured by a domestic medicine company, in the treatment of ascites inliver cirrhosis, we examined its effect by using rat models of liver cirrhosis made bythe first part of our research.The selected rat liver cirrhosis models were divided into6groups randomly.20%rHSA was administrated at dosing rates of1.25ml/kg wt,2.5ml/kg wt,5ml/kg wt and10ml/kg wt (0.25g/kg wt,0.5g/kg wt,1g/kg wt and2g/kg wt of albumin) through thetail vein for2days. Administered in the same manner are20%pd-HSA at dosing rateof10ml/kg wt (2g/kg wt) and simulation reagent at10ml/kg wt for control.Abdominal circumference, as index of ascites, was measured at18h after finaladministration; blood was collected through the orbital vein at18h after completion offinal administration and analyzed for serum albumin level and blood COP. Urine wascollected from initiation of first administration to18h after completion of finaladministration, and UV was calculated for the period. After these measurements weretaken, correlations between changes from baseline value were examined betweendifferent groups.Drug effects on various parameters were assessed on the basis of change frombaseline values. The data obtained are expressed as mean SEM. Student's t-test wasused for two group comparison, and one way analysis of variance is use for multiplegroup comparison. Correlation was examined by Pearson's correlation coefficient test.The significance level was set at less than5%.rHSA decreased the abdominal circumference and UV, and increase the serumalbumin level as well as blood COP from the baseline values in a nearlydose-dependent manner, with significant positive correlation between serum albuminand COP level and negative correlation between abdominal circumference andCOP/UV. The changes are statistically insignificant between rHSA and pd-HSA group;however, changes in these treatment groups were statistically significant when compared with saline group. These findings suggested that rHSA could be effective asa therapeutic drug for ascites in patients with liver cirrhosis accompanied byhypoalbuminemia.(3) Effectiveness of pd-CSA in the treatment of ascites in rat liver cirrhosismodelsIn the third part of this study, we started a research aimed at filling the caninealbumin blank by using cold ethanol method to develop pd-CSA as a clinicalpharmacectic. We use the established the liver cirrhosis rat models accompanied withascites and hypoalbuminemia to investigate the pharmacodynamics of pd-CSA in thetreatment of ascites in liver cirrhosis in vivo.Four groups of the selected animals were administered10%pd-CSA at dosingrates of2ml/kg wt,4ml/kg wt,8ml/kg wt and16ml/kg wt (0.2g/kg wt,0.4g/kg wt,0.8g/kg wt and1.6g/kg wt); the negative control and positive control were receivedphysiological saline and10%plasma-derived human serum albumin as a dose of16ml/kg respectively. The time node and method of parameter measurement were thesame as the part two of our research.As a result, pd-CSA promoted the serum albumin concentration, COP level andUV from the baseline values in a dose dependent manner; changes were statisticallysignificant when these markers compared with saline group. Abdomen circumferencechanges showed a significant dose dependent and decrease when compared withsaline group. Meanwhile, liner correlations between albumin concentration and COPor abdomen circumference were confirmed. The findings suggest that pd-CSA couldbe a promising therapeutic drug for treating liver cirrhosis canine patients companiedwith hypoalbuminemia and ascites retention.